Project Summary/Abstract Cancer is the second deadliest disease in the United States, killing more than 500,000 Americans annually. This year, another 1.5 million will be diagnosed with one of nearly 200 different cancer types. Despite an ever---growing understanding of the environmental risk factors, genetic contributions, and tumorigenic mechanisms, the diagnoses and treatments for this disease remain inadequate. Much of what is known about cancer has come from studies of cells in culture and small animal models. While these model systems have been extremely informative, they also possess limitations and present challenges to translating promising therapies to the clinic. The lack of a large animal model that accurately replicates human cancer has been a major barrier to the development of effective diagnostic tools, interventions, and therapies for this deadly disease. Pigs share many similarities with humans in anatomy, physiology, genetics, and importantly, size. While naturally occurring tumors are rarely seen due to standard pork production practices, spontaneous and induced cancers have been studied in pigs and are highly representative of what is seen in humans. However, the extended timeframe required for inducing cancer in pigs and the accompanying phenotypic variability limit their usefulness. Our objective is to create a genetically engineered pig in which tumorigenesis can be conditionally induced in any tissue. We intend to accomplish this by mutating two of the most commonly affected genes in human cancer, KRAS and TP53 (encoding p53). KRAS is in oncogene encoding a small GTPase that couples receptor activation and downstream effectors to control cell proliferation, differentiation and survival. Activating mutations in KRAS are common in many human tumors. Known as the guardian of the genome, p53 is a transcription factor that regulates critical cell functions including cell---cycle arrest and apoptosis. The loss of proper p53 function predisposes cells to unregulated growth, tumor formation, and metastasis. Mouse models expressing conditional mutations in KRAS and TP53 have yielded excellent models representative of human cancers of the lung, pancreas, colon, and other tissues. We hypothesize that mutations in porcine KRAS and TP53 will produce similar results in pigs. Therefore, the ultimate goal of this project is to develop and commercialize KRAS/TP53--mutated pigs to serve as a platform for models of human cancer. We intend to accomplish this by combining gene targeting and somatic cell nuclear transfer. This proposal specifically outlines the development of porcine fibroblasts with mutated KRAS and TP53 alleles. A gene targeting vector will be developed and used to disrupt the endogenous porcine KRAS via homologous recombination in both TP53---targeted and wild---type cells. Subsequent work will use these cells as nuclear donors for somatic cell nuclear transfer to produce KRAS/TP53---mutated pigs. These models will provide academic and industry researchers with an opportunity to better understand cancer and its pathogenesis and to develop and test new diagnostic, therapeutic, and preventative strategies.